LOCUS       sequence-364643-        9226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of gRNA and Cas9 from S. pyogenes with 2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9226)
  AUTHORS   Asimi V, Sampath Kumar A, Niskanen H, Riemenschneider C, Hetzel S, 
            Naderi J, Fasching N, Popitsch N, Du M, Kretzmer H, Smith ZD, 
            Weigert R, Walther M, Mamde S, Meierhofer D, Wittler L, Buschow R, 
            Timmermann B, Cisse II, Ameres SL, Meissner A, Hnisz D
  TITLE     Hijacking of transcriptional condensates by endogenous retroviruses.
  JOURNAL   Nat Genet. 2022 Aug;54(8):1238-1247. doi: 
            10.1038/s41588-022-01132-w. Epub 2022 Jul 21.
  PUBMED    35864192
REFERENCE   2  (bases 1 to 9226)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41588-022-01132-w"; journalName: "Nat Genet"; date: 
            "2022-08"; volume: "54"; issue: "8"; pages: "1238-1247"
COMMENT     Sequence Label: pX458-MMETn-del-gRNA2
FEATURES             Location/Qualifiers
     source          1..9226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          102..329
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      341..350
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             350..415
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             422..442
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             467..4567
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             467..4567
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4568..4615
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4631..4684
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(4685..4687,4688..5398)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4685..4687,4688..5398)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4988..5007)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5335..5356
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5426..5443)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5432..5639
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5648..5788
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5648..5777
                     /label=AAV2 ITR
     rep_origin      5863..6318
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5950..5969)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6160..6181
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6335..6354)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6454..6476
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6514..6532)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6600..6704
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6705..6773,6774..7565)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6923..6942)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7736..8324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8225..8244
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        8386..8634
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     8386..8406
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     8557..8576
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        8656..8731
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        8828..9113
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc
       61 ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgcgctgcc
      121 ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg
      181 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagctga
      241 gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt aattacctgg
      301 agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg actataagga
      361 ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg ataagatggc
      421 cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca agaagtacag
      481 catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg acgagtacaa
      541 ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca tcaagaagaa
      601 cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc ggctgaagag
      661 aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc aagagatctt
      721 cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag agtccttcct
      781 ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg tggacgaggt
      841 ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg tggacagcac
      901 cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca agttccgggg
      961 ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca agctgttcat
     1021 ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg ccagcggcgt
     1081 ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg aaaatctgat
     1141 cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg ccctgagcct
     1201 gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca aactgcagct
     1261 gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg gcgaccagta
     1321 cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga gcgacatcct
     1381 gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca agagatacga
     1441 cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc tgcctgagaa
     1501 gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca ttgacggcgg
     1561 agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga tggacggcac
     1621 cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc ggaccttcga
     1681 caacggcagc atcccccacc agatccacct gggagagctg cacgccattc tgcggcggca
     1741 ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga tcctgacctt
     1801 ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg cctggatgac
     1861 cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg acaagggcgc
     1921 ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc ccaacgagaa
     1981 ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg agctgaccaa
     2041 agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg agcagaaaaa
     2101 ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc agctgaaaga
     2161 ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg tggaagatcg
     2221 gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg acaaggactt
     2281 cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc tgacactgtt
     2341 tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt tcgacgacaa
     2401 agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga gccggaagct
     2461 gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc tgaagtccga
     2521 cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga cctttaaaga
     2581 ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc acattgccaa
     2641 tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg tggtggacga
     2701 gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa tggccagaga
     2761 gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc ggatcgaaga
     2821 gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa acacccagct
     2881 gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt acgtggacca
     2941 ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc ctcagagctt
     3001 tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga accggggcaa
     3061 gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact ggcggcagct
     3121 gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg ccgagagagg
     3181 cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg aaacccggca
     3241 gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt acgacgagaa
     3301 tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg tgtccgattt
     3361 ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc acgcccacga
     3421 cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta agctggaaag
     3481 cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg ccaagagcga
     3541 gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca tgaacttttt
     3601 caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga tcgagacaaa
     3661 cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg tgcggaaagt
     3721 gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag gcggcttcag
     3781 caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa agaaggactg
     3841 ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg tgctggtggt
     3901 ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc tgctggggat
     3961 caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg aagccaaggg
     4021 ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc tgttcgagct
     4081 ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg gaaacgaact
     4141 ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg agaagctgaa
     4201 gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca agcactacct
     4261 ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg ccgacgctaa
     4321 tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca gagagcaggc
     4381 cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg ccttcaagta
     4441 ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc tggacgccac
     4501 cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt ctcagctggg
     4561 aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaaggaatt
     4621 cggcagtgga gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg
     4681 cccagtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     4741 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     4801 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     4861 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     4921 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     4981 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     5041 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     5101 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     5161 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     5221 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     5281 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     5341 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagga
     5401 attctaacta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     5461 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     5521 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     5581 ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag
     5641 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     5701 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     5761 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     5821 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     5881 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     5941 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     6001 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     6061 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     6121 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     6181 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     6241 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     6301 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6361 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6421 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     6481 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     6541 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     6601 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6661 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6721 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6781 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6841 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6901 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6961 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     7021 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     7081 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     7141 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7201 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7261 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     7321 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7381 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     7441 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7501 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7561 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7621 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7681 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7741 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7801 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7861 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7921 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7981 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     8041 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     8101 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     8161 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     8221 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     8281 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     8341 cctttttacg gttcctggcc ttttgctggc cttttgctca catgtgaggg cctatttccc
     8401 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat
     8461 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     8521 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact
     8581 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgttgct
     8641 gtggcaaaca tgtgagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     8701 tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag
     8761 ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag
     8821 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     8881 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     8941 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     9001 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
     9061 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
     9121 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
     9181 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggg
//
